Soluble guanylyl cyclase subunits act as Hsp90 co-chaperones to ensure the expression and functional maturation of hemeproteins in mammalian cells

Abstract

The cofactor Fe-protoporphyrin IX cofactor (heme) performs many functions in biology. Animal cells must stabilize their newly generated heme-free (apo)-hemeproteins and deliver mitochondrial heme to them so they can mature to functional form. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) typically accomplishes the heme deliveries, and for many apo-hemeproteins, heat shock protein 90 (Hsp90) drives their heme insertions. We previously observed hemeproteins express poorly in a cell line (COS-7) that does not express soluble guanylyl cyclase (sGC), a heme-binding enzyme that typically functions through its cGMP generation. To understand sGC involvement, we expressed four hemeproteins, Hemoglobin beta (Hb{beta}), Myoglobin (Mb), Indoleamine 2,3-dioxygenase 1 (IDO1), and Tryptophan 2,3-dioxygenase (TDO) in a cell line expressing sGC (HEK293) or in two cell lines (COS-7, DU145) that do not. We assessed hemeprotein expression levels, their abilities to acquire heme, and when relevant if these facets could be rescued by co-expressing individual sGC subunits, including variants with defects in either sGC heme binding, Hsp90 association, heterodimerization, or cGMP production. We found that co-expression of either sGC subunit was essential for three of the four apo-hemeproteins to accumulate in the COS7 and DU145 cells and acquire heme. This did not involve heme binding, heterodimer formation, or cGMP generation by the sGC subunits, and instead depended on a subunits ability to recruit Hsp90 and GAPDH to the apo-hemeproteins via their own Hsp90 binding. Recruiting Hsp90 and GAPDH to apo-hemeprotein clients to ensure they can accumulate and mature to functional form broadens our understanding of sGC and Hsp90 functions in biology.